Photosensitive Hydrogel with Temperature‐Controlled Reversible Nano‐Apertures for Single‐Cell Protein Analysis

Abstract Single cell western blot (scWB) is one of the most important methods for cellular heterogeneity profiling. However, current scWB based on conventional photoactive polyacrylamide hydrogel material suffers from the tradeoff between in‐gel probing and separation resolution. Here, a highly sensitive temperature‐controlled single‐cell western blotting (tc‐scWB) method is introduced, which is based on a thermo/photo‐dualistic‐sensitive polyacrylamide hydrogel, namely acrylic acid‐functionalized graphene oxide (AFGO) assisted, N‐isopropylacrylamide modified polyacrylamide (ANP) hydrogel. The ANP hydrogel is contracted at high‐temperature to constrain protein band diffusion during microchip electrophoretic separation, while the gel aperture is expanded under low‐temperature for better antibody penetration into the hydrogel. The tc‐scWB method enables the separation and profiling of small‐molecule‐weight proteins with highly crosslinked gel (12% T) in SDS‐PAGE. The tc‐scWB is demonstrated on three metabolic and ER stress‐specific proteins (CHOP, MDH2 and FH) in four pancreatic cell subtypes, revealing the expression of key enzymes in the Krebs cycle is upregulated with enhanced ER stress. It is found that ER stress can regulate crucial enzyme (MDH2 and FH) activities of metabolic cascade in cancer cells, boosting aerobic respiration to attenuate the Warburg effect and promote cell apoptosis. The tc‐scWB is a general toolbox for the analysis of low‐abundance small‐molecular functional proteins at the single‐cell level.

Optical images of the contraction and swelling performance of NIPAM-hydrogel with different doping concentrations (0.5%-2%) under different controlled temperature conditions, continuous measurements from 0-4 hours at 1h intervals.During the duration of 4 h, the gels expand slowly under 4 °C.While under the temperature condition of 55 °C, the gels contract.The effects of temperature-modulated expansion or contraction are more pronounced at higher NIPAM doping concentrations.The FT-IR spectra of GO showed several peaks such as the -OH group at 3402.38 cm -1 , -C=O group at 1,728.91 cm -1 , the aromatic C=C skeletal vibrations at 1624.61 cm -1 , and C-O-C/C-OH vibrations at 1,000-1,400 cm -1 , which were in accordance with the literature data. 1,2 hese results indicate that abundant oxygen-containing functional gro existists on GO.After functionalizing with acrylic acid, the OH group peak at 3402.38 cm−1 faded but the -C=O group peak at 1,728.91 cm -1 enhanced.Besides, the AFGO has a characteristic =C-H peak at 2955.76 cm -1 and a characteristic -C=C-peak at 840.64 cm -1 , indicating that a portion of -OH groups have converted into -O-CO-CH=CH 2 (Supplementary Figure S7 B).The characteristic UV-Vis spectrum peak of GO significantly declined after the modification (Supplementary Figure S7 C and D).

Figure S2 .
Figure S2.MAP-mPyTC immobilizes the protein in situ with UV excitation

Figure S4 .
Figure S4.Contraction and swelling performance of the NIPAM-hydrogels

Figure S6 .
Figure S6.Mechanical performance characterization of hydrogels with different doping conditions

Figure S8 .
Figure S8.Dispersion performance of GO and AFGO in gels.Optical images of GO and AFGO dispersed in DMF and images of GO-hydrogels and AFGO-hydrogels.

Figure S9 .
Figure S9.Contraction and swelling performance of the ANP hydrogel

Figure S11 .
Figure S11.Comparison of proteins electrophoretic separation in non-ANP hydrogel (hydrogels without AFGO or NIPAM doping) and ANP hydrogel

Figure S12 .
Figure S12.The photo capture protein performance of the ANP hydrogels

Figure S13 .
Figure S13.The electrophoretic migration stability experiment.(A) Electrophoretic migration behavior visual comparison and the corresponding quantitative data of electrophoretic distance in 8%T gel and 10%T gel.Electrophoresis time: 65s, voltage:120V.(B) Electrophoretic migration behavior visual comparison and the corresponding quantitative data of electrophoretic distance in different times.Voltage:120V, Data are expressed as mean ± SD. (C) Electrophoretic migration behavior visual comparison and the corresponding quantitative data of electrophoretic distance in different temperatures.Voltage:120V, electrophoresis time: 75s.(D) Electrophoretic migration behavior visual comparison and the corresponding quantitative data of electrophoretic distance in two different electric field intensities.Electrophoresis time: 75s.

Figure S14 .
Figure S14.Comparison of different antibody incubation methods

Figure S15 .
Figure S15.Comparing the diameters of microwells in 4 °C and 55 °C ANP gels

Figure S16 .
Figure S16.Gating strategy to discriminate single and live CHOP-positive cells.